Various scientific articles, patents and other publications are referred to throughout the specification. Each of these publications is incorporated by reference herein in its entirety.
Polynucleotide amplification reactions have become central techniques of molecular biology. Indeed, many current methods in molecular biology utilize as their first step an amplification reaction involving either DNA or RNA as a template.
The polymerase chain reaction (PCR) and related techniques, such as NASBA (nucleic acid sequence-based amplification), TAS (transcription-based amplification system), 3SR (self-sustained sequence replication), LAR (ligation amplification reaction, Q-beta replicase and LCR (ligase chain reaction) are all methods of polynucleotide amplification. Many of these amplification reactions utilize a polymerase enzyme or fragment of such an enzyme.
Despite their widespread use, however, these techniques are often fraught with difficulties. In many cases, the standard procedure fails to produce meaningful amplification or any amplification at all. In other instances, the amplification of the target sequence is nonspecific, meaning that its amplification is accompanied by similar amplification of non-target polynucleotide fragments (Roux, 1995, in: Dieffenbach & Dveksler, eds., PCR Primer-A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 55-66; Newton & Graham, 1994, PCR. Bios Scientific, Oxford). These problems, especially low yield, can be particularly severe for templates with high GC contents (Varadaraj & Skinner, 1994, Gene 140, 1-5; McDowell et al., 1998, Nucl. Acids Res. 26, 3340-3347).
Accordingly, improvement of amplification and stringency has been the focus of many research efforts. It has been found that various organic additives can often yield significant improvements in this regard, the most successful of the additives tested being DMSO, glycerol, polyethylene glycol, betaine and formamide (Winship, 1989, Nucl. Acids. Res. 17, 1266; Smith et al., 1990 Amplifications 5, 16-17; Weissensteiner & Lanchbury, 1996, BioTechniques 21, 1102-1108).
U.S. Pat. Nos. 5,545,539 and 5,846,716 to Miller et al. disclose a method for improving sequencing or amplification of polynucleotides that comprises including a glycine-based osmolyte, such as trimethylglycine, in the amplification or sequencing reaction mixture. Addition of this reagent was reported to be particularly advantageous in reducing the appearance of stutter bands in the amplification product.
U.S. Pat. No. 6,114,150 to Weissman et al. discloses methods and compositions for obtaining uniform amplification of nucleic acid templates with variable G+C content by adding to the reaction mixture a zwitterion and a compound that disrupts base pairing. Compounds such as betaine, monomethyl glycine, dimethylglycine and D-carnitine are disclosed as useful zwitterions; DMSO and formamide are disclosed as useful for disrupting base-pairing.
U.S. Pat. No. 6,300,075 to Preston et al. discloses a method to amplify nucleic acids that is alleged to improve the specificity of amplification of a target nucleic acid. The method comprises supplementing an amplification reaction mixture with a carrier nucleic acid and one or more magnesium salts. Addition of these materials is reported to reduce polymerase extension of non-target nucleic acids during amplification assays through a reduction in the amount of primer-dimer formation prior to raising the temperature of the amplification mixture during thermal cycling.
U.S. Pat. No. 6,261,773 to Segawa et al. discloses that the sensitivity of amplification reactions, particularly of RNA templates, may be improved by adding EDTA or a similar reagent, such as nitriotriacetic acid (NTA), uramil diacetic acid (UDA), trans-1,2-cyclohexanediaminetetraacetic acid (CyDTA), diethylenetriamine-pentaacetic acid (DTPA), ethyleneglycolbis(2-aminoethyl)ether diaminetetraacetic acid (GEDTA) or triethylenetetraminehexaacetic acid (TTHA), or salts thereof, to the reaction mixture. These compounds are reported to improve the signal-to-noise ratio of amplification reactions by significantly inhibiting the occurrence of non-specific amplification reactions.
Despite their general applicability, the performance of the currently available compounds, especially in the case of GC-rich targets, is quite unpredictable. Any given compound often fails to provide adequate improvement over the control (Baskaran et al., 1996, Genome Methods 6, 633-638).
Thus, increasing the selection of additives that are capable of improving polynucleotide amplification, especially for recalcitrant targets, would be a significant advance in the art of nucleic acid amplification. Such new additives would be of particular benefit by improving both the potency and specificity of the amplification reaction.